Eugenol is the major component of Syzygium aromaticum essential oil. Several studies demonstrate its health benefits, particularly in inflammatory diseases. In this study, we found that eugenol inhibits the production of reactive oxygen species (ROS) by human neutrophils in resting conditions and after stimulation with two different agonists PMA or fMLF. The inhibitory effect is significant and dose dependent from low concentration of 0.1 µg/mL. In addition, eugenol clearly inhibited MPO activity at the low dose of 0.05 µg/mL. In contrast, we showed that eugenol could not affect the degranulation of neutrophils and has no scavenging effect of H2O2 and O2.−. In vivo study showed that eugenol inhibited LPS-induced lung inflammation in mice and reduced the activity of matrix metalloproteinases 2 and 9 in lung homogenates.
In chronic obstructive pulmonary disease (COPD), one of lung inflammatory disorders, the oxidative stress acts as the basic etiology13. Inflammatory cells, especially neutrophils, recruited in the site of inflammation, generate a number of reactive oxygen species (ROS) via the activation of NOX214. These ROS damage the surrounding cells leading to the development of inflammatory disorders15. Thus, pharmacological inhibition of ROS overproduction can restrict inflammatory responses16. In this study, luminol-enhanced chemilunescence assays indicated that eugenol significantly decreased total (intracellular and extracellular) ROS generation induced by the protein kinase C activator, PMA or the chemotactic peptide fMLF. PMA and fMLF are two known NADPH oxidase activators via different transduction pathways17. Thus, the inhibitory effect of eugenol on ROS production suggest that it does not affect a specific pathway but it inhibits a common target such as NADPH oxidase, MPO or scavenges reactive oxygen species. Luminol-amplified chemiluninescence is dependent on several elements including superoxide anions, H2O2 and MPO release and activity18. Hence, we found that eugenol does not affect H2O2/HRPO cell-free chemiluminescence but it inhibits strongly myeloperoxidase activity at low concentration (0.05 µg/mL) without affecting its cellular release. MPO is an important member of innate immune defense and oxygen dependent microbicidal activity of phagocytes19. It is released from the azurophilic granules of activated neutrophils. Excessive release of MPO leads to the initiation and progression of various inflammatory diseases20. A recent study showed that the inhibition of MPO activity, using its pharmacological inhibitor AZM198, reduced degranulation of ANCA-stimulated neutrophils and attenuated endothelial cell damage caused by neutrophils infiltration. Besides, in a model of crescentic glomerulonephritic, MPO inhibition suppressed kidney damage without increase in adaptative T cell responses21. Thus, MPO could be a target for the treatment of inflammatory diseases. For this, there is a growing interest in the research and development of MPO inhibitors from natural molecules. A comparative study of the effect of fifteen essential oils on MPO activity in human neutrophils in vitro demonstrated that eugenol exhibits the highest inhibition of MPO activity with IC50 of 19.2 µg/mL22.
LPS is known as a potent inflammatory stimulus used to induce lung inflammation in animal models23. It activates the transmembrane receptor TLR-4 which is expressed in pro-inflammatory cells24. Activated macrophages induce an influx of inflammatory cells, especially neutrophils, which produce increasing levels of pro-inflammatory cytokines and reactive oxygen species25. ROS production leads to the activation of many signaling proteins including NF-KB, MAPKs, iNOS and COX-2 which accentuate the inflammatory response26. Recruitment of neutrophils is an important step in the development of lung injury. Thus, the inhibition of neutrophils infiltration can be a target to attenuate lung inflammation. Our results showed that eugenol reduces the increased influx of neutrophils into lung parenchyma and BALF. Furthermore, we found that eugenol down regulated total protein levels and decreased the formation of lung edema. Since reactive oxygen species are involved in inducing inflammation, results of this work can be explained in part by our previous study demonstrating clearly the antioxidant effect of eugenol as a major inhibitor of NADPH oxidase activity by inhibiting Raf/MEK/ERK1/2/ p47phox-phosphorylation pathway11. One other study has shown that a specific inhibitor of COX-2 significantly reduced neutrophil recruitment into lung adenocarcenoma A549, which was associated with slower growing tumors27. It was shown also that the neutralization of interleukine-8 (IL-8), a major chemoattractant of neutrophils, reduced significantly neutrophils count into the brochoalveolar space and protected rabbits from acid-aspiration induced lung injury28.
Matrix metalloproteinases (MMPs) are a family of endopeptidases able to degrade the denatured fibrillar collagens, elastase, and several other components of the extracellular matrix. They are produced by different cell types, notably neutrophils29. MMPs are implicated in many aspects of both physiological cellular processes and pathological mechanisms30. Tissue inhibitors of mealloproteinases (TIMPs) are considered as major endogenous inhibitors of MMPs in physiological conditions31. However, in pathological conditions exogenous MMPs inhibitors may be either synthetic or natural one are required to stop the deleterious effect of MMPs. MMPs family exhibits a structural homology between each other but also to other zinc-dependant proteases32. Accordingly, many developed inhibitors are failed due the lack of specificity. Thus, the need of high specific inhibitor able to discriminate between the homologous MMPs and ideally administrated as short-term topical treatment33. In this work, we have shown that eugenol can indeed inhibits MMP-2 and MMP-9 activity induced by LPS administration. Indeed, it was already shown that eugenol inhibited significantly the activity of MMP-9, in the concentration range of 10–100 µM, in PMA-stimulated H1080 cells via the reduction of oxidative stress parameters34. Furthermore, Abdullah et al. demonstrated that the treatment of breast cell lines MDA-231 and SK-BR-3 with eugenol at 4 µM and 8 µM for 48 hours inhibited significantly their proliferation with an inhibition rate of 76.4% and 68.1% respectively. Eugenol treated cells showed also decreased MMP-2 activity and increased proportion of cells in late apoptosis35.
Natural bioactive molecules found in medicinal plants are attractive for the development of effective drugs for several diseases especially those associated with inflammatory and oxidant disorders36. Epidemiological studies have shown that populations of consumers of fruits and vegetables rich in polyphenols, exhibiting high antioxidant proprieties, have a lower incidence of developing inflammatory diseases37, 38. As natural polyphenol, eugenol has shown a beneficial effect in a variety of inflammatory models. In fact, a Brazilian group has shown that the oral administration of eugenol, at doses of 200 mg/kg and 400 mg/Kg, reduced significantly paw edema, 2–4 hours after carrageenan injection in mice. The inhibition was comparable to each of indomethacinan and celexib, two known anti-inflammatory molecules39. Another in vitro study demonstrated that eugenol exhibits a tumor suppressive effect on lung cancer cells. It inhibits cell viability, proliferation, migration and invasion at all tested concentrations (50 µM to 1000 µM). This effect was explained by the inhibition of PI3K/AKT pathway and the reduction of MMP-2 activity, supporting the use of eugenol as chemotherapeutic agent against human lung cancer40.